Conferred susceptibility to lambda phage in non-coliform procaryotic hosts
AssigneeThe Regents of the University of California
InventorsRobert A. Ludwig, Gert E. de Vries
Life of this patent
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Abstract
Vectors suitable for effecting expression of lamB protein in a desired procaryotic host, and methods for their construction, are disclosed. Transformation with these vectors results in the ability of the procaryotic host to sustain infection by lambda phage.
Claims18
A recombinant DNA sequence which comprises the DNA sequence encoding lamB protein under control of a constitutive promoter.
A method to prepare a mutant strain, wherein the gene encoding lamB under control of a constitutive E. coli promoter which method comprises:
- (a) selecting a mutant strain of E. coli which is resistant to lambda phage;
- (b) selecting a revertant of the mutant strain of (a) which is sensitive to lambda infection;
- and
- (c) confirming the constitutive production of lamB in said revertant strain by confirming lambda sensitivity on maltose-free medium.
A method to obtain a DNA sequence capable of constitutive expression of lamB in a non-coliform procaryotic host which comprises:
- (a) selecting constitutive lamB producing E. coli cells;
- (b) excising the lamB expression system by digesting the genomic DNA of the selected cells with one or a plurality of restriction endonuclease (s);
- (c) cloning the DNA digest from (b) into a broad host range vector to obtain a precursor plasmid;
- (d) cotransforming a target procaryotic host cell with the precursor plasmid of (c) and an additional narrow host range vector containing a sequence encoding a selectable marker in the target host which sequence is ligated to DNA homologous to lamB;
- (e) selecting transformants for the selectable marker;
- (f) removing the selective pressure, and screening for loss of the selectable marker;
- and
- (g) recovering said lamB expression system DNA sequence.
A method to prepare a non-coliform procaryotic host capable of lamB expression, which method comprises:
- cotransforming non-coliform procaryotic cells with
- a first plasmid containing the lamB gene under control of a constitutive promoter and a broad host range origin of replication, and
- a second plasmid containing a sequence encoding a selectable marker in the target host which sequence is ligated to DNA homologous to lamB;
- selecting the cotransformed cells for recombination between said first and second plasmids under selection pressure for the marker;
- and
- removing the selection pressure to obtain a non-coliform procaryotic colony harboring an operable expression system for lamB.
Dependencies are inferred from the claim text.